1. Field of the Invention
The present invention relates to an apparatus and method for assay in utilizing attenuated total reflection. More particularly, the present invention relates to an apparatus and method for assay in utilizing attenuated total reflection, in which a flow channel can be protected from backflow of waste fluid.
2. Description Related to the Prior Art
An assay apparatus in utilizing attenuated total reflection for assaying a sample is known in the field of the biosensor. U.S. Pat. No. 5,313,264 (corresponding to JP-A 4-501462) discloses a surface plasmon resonance (SPR) sensor as a typical example for this assay.
A thin film, or metal film, is formed on a transparent dielectric medium. One surface of the metal film is a sensing surface where reaction of a sample occurs. Another surface of the metal film is a thin film/dielectric interface where light is applied by satisfying a condition of total reflection. The reaction is detected to assay the sample according to attenuation of the reflected light from the thin film/dielectric interface. In a surface plasmon resonance (SPR) sensor, surface plasmon is a term to mean the compressional wave created on the surface of the metal and included in plasmon as quantized expression of the compressional wave. Free electrons in a metal vibrate to generate the compressional wave.
The surface plasmon resonance (SPR) assay apparatus is constructed to detect surface plasmon resonance created on the sensing surface which is a first surface of the metal film, and along which the surface plasmon travels.
Light for detection is applied to a thin film/dielectric interface of the metal film that is back to the sensing surface so that the total reflection condition is satisfied, namely at an angle of incidence equal to or more than a critical angle. In addition to the total reflection created on the thin film/dielectric interface, a small component of the light passes through the metal film without reflection, and penetrates to the sensing surface. A wave of the penetrating component is called an evanescent wave. Surface plasmon resonance (SPR) is created when frequency of the evanescent wave coincides with that of the surface plasmon. In response to this, intensity of the reflected light attenuates remarkably. In the assay apparatus, the attenuation in the reflected light reflected by the thin film/dielectric interface is detected, to recognize creation of the SPR on the sensing surface.
The angle of incidence, namely resonance angle of the light to generate the SPR depends on the refraction index of the transmission medium transmitting evanescent wave and surface plasmon. In other words, a change in the resonance angle to create SPR changes in response to a change in the refraction index of the transmission medium. The substance contacting the sensing surface is a transmission medium transmitting the evanescent wave and surface plasmon. If binding or dissociation between two molecules occurs on the sensing surface, the resonance angle changes because of a change in the refraction index of the transmission medium. In the SPR system, the change in the refraction index is detected, to measure interaction of molecules.
The assay apparatus can be used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. Also, the technique is useful in the fields of the clinical medicine, food industries and the like. It is possible to use one of two substances as a ligand and another of them as an analyte if those have bioaffinity. For the purpose of screening, protein as biomaterial is used as ligand. Candidate drugs are discretely used as analyte, and contacted with the ligand on the sensing surface, to study interaction.
JP-A 6-167443 and U.S. Pat. No. 5,822,073 disclose an SPR assay apparatus in which an optical system of Kretschmann configuration is used for incidence of light to the metal film. According to the Kretschmann configuration, the thin film/dielectric interface of the metal film is fitted on a prism, which condenses light and directs the light to the thin film/dielectric interface in a manner conditioned for total reflection. A sample or ligand is immobilized on the sensing surface. A flow channel is formed to have the sensing surface inside, and causes analyte fluid to flow. The analyte fluid is introduced in the flow channel to flow, and is caused to contact the ligand. Interaction between the analyte fluid and the ligand is assayed by detecting surface plasmon resonance created during the reaction.
At first in an assay, liquid buffer is introduced to a flow channel to contact a sensing surface. An output signal starts being measured with the buffer. Then analyte fluid is introduced. The liquid buffer in the flow channel is pushed out by the analyte fluid, and is drained through an exit end opening of the flow channel. The analyte fluid is kept to exist in the flow channel for a prescribed time, before the liquid buffer is introduced again. Then the measurement of the output signal is completed. According to detection of a base line of the output signal, detection is possible during a process starting at association between the analyte and ligand and ending at dissociation between those.
Examples of methods for introducing the analyte fluid on to the sensing surface includes delivery by means of conduits and pumps, and delivery through a pipette couple. The pipette couple is easily settable on end openings of the flow channel, and suitable for access to numerous sensor cells by loading and unloading at many times. Specifically, a dispensing pipette included in the pipette couple is set at the flow channel for dispensing the analyte fluid. A removing pipette included in the pipette couple removes the liquid buffer through the flow channel through the exit end opening by suction.
However, there is a problem in the method of the pipette couple. After the introduction, the liquid buffer from the exit end opening is likely to leak from the end of the removing pipette upon unloading of the pipette couple. Droplets of the liquid buffer will enter the exit end opening to flow back through the flow channel. A backflow of the liquid buffer causes a serious problem in electric noise in an output signal of the assay.